Genetic detoxification of pertussis toxin

ABSTRACT

A new method is described for the preparation of a safe, immunogenic and efficacious vaccine for protection against the disease pertussis. In development of this vaccine, specific functional sites of pertussis toxin have been identified, and using this information, defined mutant holotoxins have been produced by site directed mutagenesis of the toxin gene. A number of these toxin analogues are detoxified, retain an immunodominant S1 epitope, are immunogenic and are protective in the standard pertussis vaccine potency test in mice.

This is a division of application Ser. No. 375,376 filed Nov. 23, 1988(now U.S. Pat. No. 5,085,862).

FIELD OF INVENTION

The present invention relates to a novel method for the detoxificationof pertussis toxin by the genetic manipulation of DNA segments codingfor one or more amino acid residues essential for the toxin's biologicalactivity. It also relates to a procedure for the creation of geneticallyaltered Bordetella pertussis bacteria that produce the said detoxifiedpertussis toxin.

BACKGROUND OF THE INVENTION

Whooping cough, or pertussis, is a severe, highly contagious respiratorydisease of infants and young children caused by infection withBordetella pertussis. Owing to the many virulence factors associatedwith this organism, the pathogenesis of the disease is still not fullyunderstood; however, it is generally recognized that the major systemiceffects are caused by pertussis toxin (PT). This material exhibits awide range of biological activities as illustrated by such alternativenames as lymphocytosis-promoting factor, histamine-sensitizing factorand islet-activating protein. Many of these effects are associated withits biochemical function as an adenosine diphosphate(ADP)-ribosyltransferase. ADP- ribosylation of certain acceptorguanosine triphosphate-binding proteins leads to a loss of control overa variety of metabolic pathways mediated by cyclic adenosinemonophosphate and by phospholipase C. In the absence of a proteinacceptor, PT also catalyses the hydrolysis of nicotinamide adeninedinucleotide (AND glycohydrolase activity).

Conventional killed whole-cell pertussis vaccines contain a mixture ofantigens and there has been a great deal of work towards the developmentof a defined acellular vaccine comprising specific protective antigens.PT is the most significant protective antigen. Other antigens underconsideration are agglutinogens and filamentous hemagglutinin (FHA).

Normally PT and other antigens are chemically inactivated, or toxoided,using agents such as formaldehyde, glutaraldehyde or hydrogen peroxide.This approach has the serious disadvantage that a delicate balance mustbe sought between too much and too little chemical modification. If thetreatment is insufficient, the vaccine may retain residual toxicityowing to the presence of a small proportion of unchanged virulencefactors including PT. If the treatment is too excessive, the vaccine maylose potency because its native immunogenic determinants are masked ordestroyed. This problem is of particular concern in the case of PT,since the catalytic subunit is comparatively difficult to inactivate byaldehydes. The possible residual toxicity or reversion of toxoidedwhole-cell pertussis vaccines has been questioned for many years, and ithas suggested that in rare cases the vaccine might cause majorneurological damage. All pertussis vaccines that are in use at present,or in the trial stages, depend on the inactivation of the antigens bychemical means, which introduces the problems previously mentioned. Itis obvious that if an inactivated vaccine could be designed withoutresorting to the toxoiding process, but preserving the native structureof immunogenic and protective epitopes, an additional degree of safetyand efficacy would be added. For these reasons the inventors havegenetically manipulated the gene coding for PTTOX-, and constructedstrains of pertussis that secrete non-toxic PT analogues.

In its structural organization, PT belongs to the family ofADP-ribosyltransferase bacterial toxins, which also includes diphtheriatoxin, Pseudomonas aeruginosa exotoxin A, cholera toxin and Escherichiacoli heat labile toxin. Accordingly, it consists of two functionalmoieties: an A portion, which carries the enzymic activity, and a Bportion, which binds to the host cell and permits translocation of the Aportion to its site of action. In PT, the A portion is a discretesubunit, commonly denoted S1. The B portion is a non-covalent oligomerof five polypeptides arranged as two dimers, comprising subunits S2 plusS4 and subunits S3 plus S4 respectively, held together by a joiningsubunit S5.

The amino acid sequence of the S1 subunit reveals several features ofinterest. There are only two cysteine residues which form an intrachaindisulphide bond; however, it is known that for enzymic activity thetoxin must be reduced (Moss et al., J.Biol.Chem. 258, 11879, [1983]),indicating the importance of these residues. There are two tryptophansin S1, and it has been suggested that tryptophan residues are close tothe AND binding sites of diphtheria toxin and P. aeruginosa exotoxin A.Two conserved regions in S1 are also found in the amino acid sequencesof cholera toxin and E. coli heat labile toxin (Locht & Keith, Science,232, 1258, [1986]). In addition the AND active sites of diphtheria toxinand P. aeruginosa exotoxin A have been shown to contain a glutamic acidresidue (Carrol & Collier, Proc. Nat. Acad. Sci., U.S.A., 81, 3307,[1984]; Carroll & Collier, J.Biol.Chem., 262, 8707, [1987]).

As noted above, the B portion of PT mediates its binding to cellularreceptors and contains two dimers. Whether each of these dimers bears abinding site remains controversial. However, the S2 and S3 subunits aresimilar in amino acid sequence and binding studies have indicated thatlysine and/or tyrosine residues of S3 in particular are implicated inthe interaction of the toxin with its receptor. (Nogimori et al.,Biochem., 25, 1355, [1986]; Armstrong & Peppler, Infect. Immun., 55,1294, [1987]).

Site-directed mutagenesis of diphtheria toxin and P. aeruginosa exotoxinA at the AND-interacting glutamic acid residues has led to significantreduction in ADP-ribosyltransferase activity (Tweten et al.,J.Biol.Chem., 260, 10392, [1985]; Douglas & Collier, J.Bacteriol., 169,4967, [1987]). Complete truncated forms of S1 and S2 have been expressedin E. coli (Locht et al. Infect. Immun., 55, 2546, [1987]). Mutations ofthe TOX operon generated by transposon insertion, gene truncation orlinker insertion have been introduced by allelic exchange into thechromosome of B. pertussis (Black et al., Ann. Sclavo, 175, [1986];Black & Falkow, Infect. Immun., 55, 2465, [1987]). However, thebiological and immunoprotective properties of fully-assembledrecombinant holotoxins specifically detoxified by site-directedmutagenesis of functional amino acid residues have not been reported.The generation of such PT analogues for inclusion in a safe andefficacious pertussis vaccine is the subject of this invention

In testing for the efficacy and toxicity of materials that could becandidates for a protective vaccine, there are a number of in vivo andin vitro assays available. The standard test for potency is the mouseprotection test, which involves intra- cerebral challenge with live B.pertussis. Newer vaccine tests measure the production of protectiveantibodies. A common toxicity test is the CHO (Chinese hamster ovary)cell clustering assay, which reflects both the ADP-ribosyltransferaseand binding ability of the toxin (Burns et al., Infect. Immun., 55, 24,[1987]). A direct test of the enzymic activity of PT is theADP-ribosylation of bovine transducin (Walkins et al., J. Biol. Chem.,260, 13478, [1985]).

SUMMARY OF INVENTION

In accordance with the present invention, there is provided a novelmethod of detoxifying PT, which does not suffer from the drawbacks ofthe prior art chemical methods and yet provides an detoxified PT thatretains its immunological properties without possessing undesirable sideeffects. In the present invention, amino acid residues of the toxin thatare crucially important to its functional and toxic activities areidentified. These residues are subsequently removed or replaced bysite-directed mutagenesis of the isolated toxin gene. The mutated toxinoperon resulting from such manipulations then is substituted for thenative gene in the organism, which thereby produces the non-toxic analogof the toxin under normal growth conditions. In this manner, thethree-dimensional structure and thus the immunogenicity of the PTanalogue is minimally impaired. Indeed, an appropriate mutant form ofthe toxin on its own may provide satisfactory protection against thesevere symptoms of pertussis, though other components may be required toestablish resistance against the bacterial infection itself.

In accordance with one aspect of the present invention, there fore,there is provided an immunoprotective genetically-detoxified mutant ofpertussis toxin. By the term "genetically-detoxified" as used herein ismeant a pertussis toxin mutant which exhibits a residual toxicity ofabout 1% or less, preferably less than about 0.5%, of that of the nativetoxin. The residual toxicity is determined by CHO cell clustering assayand ADP-ribosyltransferase activity.

In accordance with another aspect of the present invention, there isprovided a vaccine against Bordetella pertussis comprising animmunogenically-effective amount of the immunoprotective mutant ofpertussis toxin or a toxoid thereof and a physiologically-acceptablecarrier therefor. The genetically-detoxified pertussis toxin also may beused as a carrier protein for hapten, polysaccharides or peptides tomake a conjugate vaccine against antigenic determinants unrelated to thetoxin.

A further aspect of the present invention provides a method ofproduction of the mutant, which comprises identifying at least one aminoacid residue of the toxin which confers toxicity to the toxin; effectingsite-directed mutagenesis of the toxin gene to remove or replace atleast one such residue and to produce a mutated toxin operon;substituting the mutated toxin operon for the native gene in theBordetella pertussis organism; and growing the transformed organism toproduce an immunoprotective, genetically-detoxified toxin.

As will be apparent from the following disclosure, the present inventionfurther provides novel strains of Bordetella pertussis from which thetoxin operon has been removed or has been replaced by a mutant gene asprovided herein.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the sequences of amino acids obtained by automatedsequencing of radiolabelled peptides A from subunit S1 which B and arecompared with residues from mature S1;

FIGS 2-2E show the structures of various TOX clones obtained from thechromosomal libraries;

FIG. 3 shows the construction of subclones containing the TOX gene fromthe genomic clone λ gtll 15-4-1, with the TOX gene being inserted intothe multiple cloning site of pUC8:2, which contains Bgl II and Xba Isites;

FIGS. 4(a-e) show the construction of subclones of the TOX gene used forsequencing the operon. In FIG. 4(a), a restriction map of the TOX geneand the protein subunits are indicated, with clones being derived fromthe pUC8:2/TOX clone J-169-1, and the subunit genes being subcloned intoM13mp18, M13mp19 or pUC8:2, as indicated; in FIG. 4(b), clones of the 5'region of pUC8:2, S1 in M13mp18 and S1 in M13mp19 clones are described;in FIG. 4(c), clones of S2 in M13mp18 and M13mp19 is shown; in FIG.4(d), clones of S4/S5 in M13mp18 and M13mp19 are shown; and, in FIG.4(e), clones of S3 and the 3' region in M13mp18 and pUC8:2;

FIG. 5(a-f) shows the nucleotide sequence and structural genetranslation products of the B. pertussis 10536 TOX gene;

FIG. 6(a-c) shows the construction of TOX or TOX analogue genes in thebroad-host-range plasmid pRK404 (Ditta et al., Plasmid, 13, 149,[1985]). In FIG. 6(a) and FIG. 6(b), there is shown the construction ofprimary TOX analogue genes in pRK404 from mutated genes and nativegenes, while in FIG. 6(c), there is shown a typical construction of a"crossed" mutant from two S1-mutated genes;

FIG. 7 shows the development of a "suicide" plasmid, one capable ofconjugative transfer but not replication, based on pRK404 and pMK2004(Kahn et al., Methods in Enzymology, 68, 278, [1979]), fornon-homologous recombination. The final plasmids also contain akanamycin resistance gene 3' of the TOX or TOX analogue genes;

FIG. 8(a-c) shows the cloning of the 5'- and 3' flanking region of theTOX gene. FIG. 8(a) shows the construction of the 5' portion of TOX inpUC8:2 from the

Charon 35 clone Ch421; FIG. 8(b) shows the construction of the 3'portion of TOX in pUC8:2 from λ Ch 111; and FIG. 8(c) shows thegeneration of a pUC8:2 clone containing TOX plus its 5'- and 3'-flanking regions;

FIG. 9(a-c) shows the construction of plasmids for the deletion of theTOX operon from the B. pertussis chromosome by homologous recombination;FIG. 9(a) shows construction of S-2544-2 FIG. 9(b) shows construction ofS-2832-1 and S-2832-5, FIG. 9(c) shows construction of PG 265, and

FIG. 10 shows the construction of plasmids for reintegration of TOXanalogues into the B. pertussis genome by homologous recombination, thefinal plasmids being based on the suicide plasmid shown in FIG. 7 andcontaining the tetracycline resistance gene from pRK404 placed 3' to theTOX analogue gene.

GENERAL DESCRIPTION OF INVENTION

It has been shown that the TOX operons from different strains of B.pertussis are nearly identical in sequence (Nicosia et al, Proc. Nat.Acad. Sci., U.S.A., 83, 4631, [1986]; Locht & Keith, Science, 232 1258,[1986]). The TOX locus is here defined as a DNA fragment beginning atthe EcoR I cleavage site which encodes a 5'-flanking sequence, thepromoter region, the structural genes for all PT subunits and a 3'flanking sequence. The TOX gene from B. pertussis 10536, which is thestrain used by the inventors, was cloned and sequenced Its nucleic acidsequence was found to be highly homologous to other published sequences,with four unique base differences downstream from the G of the EcoR Isite defined as base 1. The complete nucleotide and corresponding aminoacid sequences of the structural genes are shown in FIG. 5.

The plasmid DNA of clone J-169-1 which contains the TOX gene fromBordetella pertussis 10536 cloned into pUC8:2 as a 4.6 kb EcoR I, BamHIfragment, has been deposited with the American Type Culture Collection(ATCC) in Rochville, Maryland, U.S.A. on Nov. 23, 1988 under AmericanNumber 40518.

The T at position 315 is unique to strain 10536 and there are threedifferences in the S1 gene at positions 710, 1200 and 1202, resulting intwo unique amino acids, glutamic acid and valine, at positions 34 and198 of the mature S1 sequence, respectively. The toxin genes of B.parapertussis and B. bronchiseptica are not expressed because ofmultiple mutations in their promoter regions, (Arico & Rappuoli,J.Bacteriol., 169, 2849, [1987]). This has allowed the use of B.parapertussis as a host for the expression of mutated toxin genes forscreening purposes.

The inventors have shown that substitution of a single amino acid in S1,in particular at the active site for AND hydrolysis (position 129),virtually abolishes the ADP-ribosyltransferase activity of PT. However,it may be desirable to alter several sites on the holotoxin to ensurecomplete safety. Accordingly, this invention applies to single ormultiple mutations in both or either of the A and B portions of thetoxin to abolish toxicity, and to the reinsertion of these mutationsback into the genome of Tox⁻ strains of Bordetella.

A number of strategies have been used by the inventors to determineregions of the toxin that might be closely associated with itsbiological activities, and might, therefore, contain candidate sites forgenetic manipulation.

PT was prepared from culture supernatants of B. pertussis (strain10536). The crude solution was concentrated by ultrafiltration andpassed through a fetuin-agarose affinity column to adsorb PT. PT waseluted from the washed column using potassium thiocyanate and dialyzedinto a phosphate-saline medium. At this stage, the purity was 90-95%, asdetermined by sodium dodecyl sulphate - polyacrylamide gelelectrophoresis (SDS-PAGE) analysis. The major contaminant was FHA.Further purification was achieved by chromatography through ahydroxyapatite column, giving a material with a purity >99%.

The site of interaction of the S1 subunit with AND was determined byphoto-crosslinking AND to isolated and purified S1 using [¹⁴ C]AND,labelled either in the nicotinamide carbonyl group or the adeninemoiety. Radiolabel was efficiently transformed from the nicotinamidemoiety into the protein. The protein was then digested with trypsin andchromatographed on an HPLC column, giving two major radioactivepeptides. After purification the two tryptic peptides were sequencedwhich demonstrate that the first fifteen residues corresponded toresidues 118-132 of mature S1. In both the peptides, radioactivity wasassociated with an unidentified amino acid corresponding to position 129in mature S1. Radioactivity was not detected in any other position. Thisestablished that GLU¹²⁹ is the site of photo-crosslinking of AND and istherefore likely to be an important component of the nicotinamideinteraction site. Significantly the sites of linkage in diphtheria toxinand P. aeruginosa exotoxin A are also glutamic acid residues and thethree amino acid sequence commencing at GLU¹²⁹ of S1 resembles theanalogous sequences of the other bacterial toxins.

Chromosomal DNA was prepared from B. pertussis (strain 10536) and wasdigested with the restriction enzyme EcoR I in such a way that fragmentswere obtained ranging in size from a few hundred bases to a fewkilobases. The DNA fragments were ligated with λ gtll DNA which had beendigested with EcoR I and dephosphorylated. The DNA was packaged intophage particles and maintained in E. coli Y1090 as a gtll B. pertussisgenomic library. Alternatively, B. pertussis chromosomal DNA wasdigested with the restriction enzyme Sau3A I to generate very large DNAfragments which were ligated with BamH I restricted λ Charon 35 DNA. TheDNA was packaged into phage particles and maintained in E. coli LE392 asa λ Ch 35 B. pertussis genomic library.

These genomic libraries were plated and phage plaques transferred ontonitrocellulose filters. The filters were screened by DNA hybridizationusing an oligonucleotide probe specific for the PT S4 subunit. Positiveplaques were further purified by two additional rounds of plating andhybridization. Phage DNA was prepared from the positive plaques andsubjected to restriction enzyme digestion and Southern blot analysisClones containing the entire 4.6 kb EcoR I pertussis toxin operon (TOX)or portions thereof and with differing 5'-or 3'-flanking regions werecharacterized. The TOX gene was subcloned for sequence analysis andfurther genetic manipulation Sequencing was performed using the dideoxychain termination method and the results indicated four novel bases inthe 10536 TOX gene as compared to published sequences.

Subclones of S1 or S3 genes in M13 phage were subjected to in vitrosite-directed mutagenesis using the phosphorothioate procedure.Single-stranded DNA from these clones was annealed with oligonucleotideprimers specifically designed to mutate or delete one or more aminoacids The mutagenesis was carried out using a kit available from acommercial source. Mutations were verified by sequencing ofsingle-stranded phage DNA. Mutant subunit genes were recombined with theremainder of the operon to construct mutant holotoxin genes in thebroad-host-range plasmid pRK404 maintained in E. coli JM109.

In order to characterize the holotoxin analogues, these plasmids weretransferred to a spontaneous streptomycin-resistant B. parapertussisstrain by conjugation on a solid surface, using pRK2013 as a helperplasmid. The colonies were selected on streptomycin andtetracycline-containing Bordet-Gengou blood plates. Mutated genes werealso integrated into the chromosome of B. parapertussis by conjugativetransfer of a suicide plasmid. The integration was either random ordirected through homologous recombination utilizing the flanking regionsof the B. pertussis TOX operon. FIG. 7 shows the construction of asuicide plasmid containing mutants for random recombination.

Liquid cultures were grown in modified Stainer-Scholte medium containingmethyl-β-cyclodextrin in shake flasks (10 ml to 2L) or in fermentors(20L to 50L). The expression level of holotoxin analogues in culturesupernatants was determined by enzyme-linked immunosorbent assay (ELISA)and found to vary with the mutation. The residual toxicity of theanalogues was measured by the CHO cell clustering assay.

A number of PT analogues were purified from 2L to 50L cultures ofrecombinant B. pertussis strains, according to methods described indetail for native PT. The ADP-ribosyltransferase activity of thesemutants was determined as the extent of incorporation of radioactivityinto bovine transducin from [³² P]-labelled AND. Table la below liststhe PT mutants generated and Table 1b below summarizes their residualtoxicity and enzymic activity.

Selected purified mutants were tested in mice for acute toxicity,histamine sensitization activity and potency in the standard mouseintracerebral challenge test. These results are presented in Table 2below and show that PT analogues have a markedly-decreased acutetoxicity and histamine sensitization activity and that they areimmunoprotective in the mouse potency test.

The immunological properties of PT analogues were further investigatedby epitope mapping and by analysis of the antibody response in mice.Several monoclonal antibodies (MAbs) specific for individual subunits ordimers of PT were prepared and used to determine by ELISA whether theepitopes defined by these antibodies were affected by the mutations. TheS1 epitope recognized by MAb PS21 is of particular significance, sinceit is immunodominant in mice and this antibody confers passiveprotection in the mouse intracerebral challenge test. (The hybridomawhich secretes the monoclonal antibody PS21 has been deposited with ATCCon Nov. 30, 1989 under accession number HB 10299). The preservation ofthis epitope in the PT analogues is indicated in Table 1b.

Immunogenicity studies in mice were performed on three purified PTmutants. Immune sera were tested for their ability to inhibit PT-inducedCHO cell clustering (Table 3 below), and for their anti-PT, anti-SI andanti-B-oligomer antibody titres by indirect ELISA (Table 4 below).

To generate a B. pertussis strain expressing a mutated TOX gene suitablefor vaccine production, the endogenous TOX operon was deleted byhomologous recombination using electroporation of linear B. pertussisDNA containing the 5'- and 3'-flanking regions of the TOX locus.Selected mutant genes were then reintegrated into the TOX locus of theB. pertussis chromosome. Clones containing mutated TOX genes were grownand the culture supernatants assayed for level of expression of PTanalogues and their residual toxicity as previously described. Theseresults are shown in Table 5 below.

Bordetella pertussis strains wherein the TOX gene has been removedentirely or has been replaced by certain clones, have been depositedwith ATCC on Nov. 23, 1988, as follows:

    ______________________________________                                                                     ATCC                                                                          Accession                                        Strain          Modification Number                                           ______________________________________                                        B. pertussis 29-9                                                                             TOX deleted  53838                                                            (Tox.sup.-)                                                   B. pertussis S-2962-1-2                                                                       S1:GLY.sup.129                                                                             53837                                            B. pertussis S-2962-2-1                                                                       S1:GLN.sup.129                                                                             53836                                            B. pertussis S-3036-2                                                                         S1:GLU.sup.58                                                                              53835                                            B. pertussis S-3122-3-1                                                                       SA:ALA.sup.41                                                                              53834                                            B. Pertussis S-3122-2-3                                                                       S1:GLY.sup.129,                                                                            53833                                                            S3:ASN.sup.92 ARG.sup.93                                      ______________________________________                                    

The TOX⁻ strain is a novel strain of Bordetella pertussis from which thetoxin operon has been removed and from which foreign DNA is absent andwhich is capable of being grown in the absence of antibiotics to produceB. pertussis antigens free of pertussis toxin.

Each of the transformed strains is a strain of Bordetella pertussis inwhich the toxin operon has been replaced by a mutant gene formed bysite-directed mutagenesis of at least one specific amino acid residueresponsible for pertussis toxin toxicity.

The data presented herein demonstrate that the inventors have produced aseries of pertussis toxin analogues that exhibit a substantial reductionin CHO cell clustering and enzymic activities (0.1 to 1% of thewild-type activity). Many of these analogues also maintain animmunodominant S1 epitope recognized by a protective monoclonalantibody. Moreover, certain of these mutants have been shown to protectmice against challenge with virulent B. pertussis at does that exhibitminimal toxicity While the majority of these results have been generatedusing PT mutants secreted by B. parapertussis, it is evident thatequivalent products are obtained by genetic manipulation of B. pertussisitself. This disclosure, therefore, presents a number of detoxifiedimmunogenic forms of pertussis toxin that would be candidates forinclusion in a novel pertussis vaccine, and a method for producing themin B. pertussis.

EXAMPLES

Methods of molecular genetics, protein biochemistry and fermentation andhydridoma technology used but not explicitly described in thisdisclosure and these Examples are amply reported in the scientificliterature and are well within the ability of those skilled in the art.

EXAMPLE I

This Example illustrates the preparation and purification of PT.

Culture supernatants of B. pertussis (strain 10536) were concentrated20-50 times by ultrafiltration through a 10,000 or 20,000 molecularweight cut-off membrane using a Millipore Pellicon cassette system. Thetoxin was adsorbed from crude concentrates by passage through afetuin-agarose affinity column equilibrated with 1 M potassium phosphate, 10 mM NaCl at pH 7.5. The volume of adsorbent was typically 1 ml permg of toxin. The loaded column was washed with 100 mM potassiumphosphate, 1 M NaCl at pH 7.5, then eluted with the same buffercontaining 3 M potassium thiocyanate to desorb the toxin. Pooledfractions were dialyzed against 50 mM Tris-HCl, 200 mM NaCl containing10% v/v glycerol at pH 8.0, to remove thiocyanate, then against 50 mMTris-HCl, 200 mM NaCl containing 50% v/v glycerol at pH 8.0, to allowstorage of the product at -200C. The yield as determined by ELISA wastypically 90-95 %. The purity as determined by SDS-PAGE was 90-95%, themajor contaminant being FHA. For further purification the stored toxinwas diluted five-fold with water and loaded onto a hydroxyapatite columnof volume 1 ml per mg of toxin, that had been equilibrated with 10 mMpotassium phosphate at pH 8.0. The column was washed with 30 mMpotassium phosphate at pH 8.0 then eluted with 100 or 200 mM potassiumphosphate to desorb the toxin Pooled fractions were dialyzed against 100mM potassium phosphate containing 50% v/v of glycerol at pH 8.0 and thefinal product stored at -200° C. The yield was typically 90-95%, and thepurity >99% as shown by SDS-PAGE.

EXAMPLE II

This Example illustrates the preparation of PT subunit S1.

PT was adsorbed to fetuin-agarose as described in Example I, then thecolumn was washed with CHAPS buffer (500 mM urea, 50 mM potassiumphosphate, 100 mM NaCl and 1% w/v ofCHAPS(3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulphonate) at pH7.5). The column was eluted with the same medium containing 500 μM ofadenosine triphosphate (ATP). The S1 subunit emerged as a sharp peak atthe column volume. The pooled fractions were passed through a cleanfetuin-agarose column equilibrated with CHAPS/ATP buffer to removeresidual B oligomer, then dialyzed against 100 mM potassium phosphatecontaining 50% v/v glycerol at pH 8.0 for storage at -200C. S1 wasquantified by reverse-phase HPLC on a Vydac C4 column by comparison ofthe integrated peak area with that of a PT standard. The yield wastypically only 20-25%, but the product was free of other subunits asdemonstrated by both SDS-PAGE and reverse-phase HPLC.

EXAMPLE III

This Example illustrates the photocrosslinking of AND to the S1 subunit.

Reaction mixtures (100 μl) containing 50 μg/ml of S1, 10 mMdithiothreitol and 50 μM AND in CHAPS buffer were placed in the wells ofa 96-well microtitre plate set in ice, preincubated for 30 min and thenirradiated at 254 nm for periods up to 3 hr at a distance of 5 cm with a9 W mercury lamp. Samples were then assayed for residual ANDglycohydrolase activity. The enzyme activity of S1 was completelyabolished after irradiation for 2 hr, whereas the extent ofphotoinactivation was only 40% under the same conditions but in theabsence of AND. This result indicated that AND-dependent photochemicalevents had occurred. To discover which part of the AND moleculeinteracted with the protein and the extent of crosslinking, S1 wasirradiated under identical conditions with [carbonyl-¹⁴ C]AND or[adenine-¹⁴ C]AND. Aliquots were removed at intervals up to 3 hr andtreated with trichloroacetic acid (TCA) to 10% w/v. The precipitatedprotein was collected by filtration, washed with fresh 10% w/v TCA andcounted in a scintillation counter. Results indicated that theradiolabel was incorporated from the nicotinamide moiety rather thanfrom the adenine moiety, and that the extent of incorporation was 0.75mol label per mol protein.

EXAMPLE IV

This Example identifies the site of photocrosslinking on the S1 subunit.15 Reaction mixtures (3 ml) containing 100 μg/ml of S1, 10 mMdithiothreitol and 50 μM [carbonyl-¹⁴ C]AND in CHAPS buffer were placedin a Petri dish on ice to give a 1 mm layer, then irradiated at 254 nmfor 2 hr with gentle magnetic stirring. The solution was deaerated withnitrogen, further reduced with dithiothreitol and S-alkylated with4-vinylpyridine to prevent oxidation of thiol groups. The reactionmixture was dialyzed extensively against 10 mM acetic acid and theradiolabelled protein was collected after precipitation with 20% w/vTCA.

The precipitated protein (1 mg) was redissolved in 2 M urea, 200 mMammonium bicarbonate to 500 μg/ml and digested with 50 μg/ml trypsin for20 hr at 37° C. The mixture was acidified and fractionated on a 1×25 cmVydac C₁₈ reverse-phase HPLC column, using a linear gradient of 0-50%acetonitrile in 10 mM trifluoracetic acid (TFA). Fractions were checkedby scintillation counting, which revealed two major radioactivepeptides, denoted A and B, accounting for 50% of the elutedradioactivity. The peptide pool was lyophilized, redissolved in 10 mMTFA, 6 M guanidinium chloride and separated on a Vydac CI8 column usinga 20-30% acetonitrile gradient in 10 mM TFA. Each peptide was furtherpurified to homogeneity on the same column by applying an acetonitrilegradient in 20 mM ammonium acetate at pH 6.5, and the solutionsevaporated to dryness. Their specific radioactivities were consistentwith only one labelled site per molecule.

The two peptides were sequenced by automated Edman degradation. Aportion of the sequenator effluent was diverted for monitoring ofradioactivity. The results are shown in FIG. 1. Up to cycle 15, thesequences proved to be identical and correspond unequivocally toresidues 118-132 of mature S1. In both peptides radioactivity wasassociated with an unidentified amino acid released at cycle 12,corresponding to position 129 in mature S1. No radioactivity wasdetected at cycles beyond 15. Thus it was established that GLU¹²⁹ wasthe site of crosslinking, and is therefore likely to be an importantcomponent of the nicotinamide interaction site.

EXAMPLE V

This Example illustrates the preparation of B. pertussis chromosomalDNA.

Two litres of B. pertussis (strain 10536) were grown in modifiedStainer-Scholte medium as 16×125 ml aliquots using a 4 ml inoculum ofsaturated growth for each flask. This medium consists of L-proline 5g/L,NaCl 2.5 g/L, KH₂ PO₄ 0.5 g/L, KCl 0.2 g/L, MgCl₂.6H₂ O 0.1 g/L, Tris1.5 g/L, casamino acids 10 g/L, methyl-β-cyclodextrim 2 g/L, CaCl₂.2H₂ O0.02 g/L, mono-sodium glutamate 10 g/L, L-cysteine 0.004%, FeSO4.7H₂ O0.001%, niacin 0.004%, glutathione 0.015%, and asorbic acid 0.04%,pH.7.6. Samples were grown in 500 ml flasks, on a shaker at 35°-36° C.,150 rpm for 16.5 hr to log phase. The cells were spun in 50o ml aliquotsat 5000 xg for 1 hr at 4° C. Each aliquot was washed with 25 ml TEbuffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5,) then resuspended in 20 ml TEand frozen at -70° C. One pellet was resuspended in 90 ml TE and pronaseadded to 500 μg/ml. SDS was added to 1% and the sample incubated at 37°C. for 21.5 hr generating a clear lysate. The lysate was extracted with1 volume of phenol saturated Tris-HCl at pH 7.5 at room temperature for2 hr, with gentle agitation. The phases were separated by centrifugationat 2800 xg for 15 min at 20° C. and the aqueous phase extractedsimilarly with 1 volume of 1:1 phenol:chloroform. The phases wereseparated by centrifugation at 2100 xg for 10 min at 20° C. and theaqueous phase extracted with chloroform for 2 hr as described. Thephases were separated by centrifugation at 1600 xg for 5 min at 20° C.and the aqueous phase subjected to dialysis at 4° C. against 2 L of 1 MNaCl for 24 hr with one change of buffer, then against 2 L TE for 48 hrwith one change of buffer.

EXAMPLE VI

This Example illustrates the generation of B. pertussis gene libraries.

1) λ gtll EcoR I library

B. pertussis DNA (10 μg) was digested with EcoR I (10 units) in thepresence of 100 mM Tris-HCl pH 7.5, 50 mM NaCl, 5 mM MgCl₂, 100 μg/mlBSA, 1 μg/ ml RNAse A for various lengths of time in order to generate aset of partially digested DNA fragments. At each time point of 0.25,0.5, 1, 2, 4 and 8 hrs, the sample was placed at 0° C. and EDTA added to20 mM to stop the reaction. The samples were pooled and separated on a10-40% sucrose gradient in TNE (20 mM Tris-HCl, pH 8.0, 5 mM EDTA, 1MNaCl) at 85,000 xg for 20 hr at 20° C. The gradient was fractionatedfrom the top as 24 aliquots (0.5 ml) to which 1 ml aliquots of absoluteethanol were added to precipitate the DNA. The samples were incubated ondry ice for 30 min then centrifuged at 12,000 xg for 5 min at 4° C. Thepellets were washed with 750 μl of 70% ethanol, incubated on dry ice for5 min, centrifuged at 12,000 xg for 5 min, then dried. Each pellet wasresuspended in 25 μl of sterile water and 5 μl aliquots of everyalternate fraction were submitted to agarose gel electrophoresis todetermine the size of the fragments. Samples containing DNA ranging insize from approximately 0.5 kb to 9 kb were pooled. The pooled EcoRI-digested B. pertussis DNA (0.4 μg) was ligated with EcoR I-digested,dephosphorylated λ gtll DNA (0.5 μg) and was packaged into phageparticles using a commercial kit. The phage library was propagated in E.coli Y1090 cells and was titred at approximately 10¹⁰ plaque-formingunits(pfu)/λg of λ gtll DNA. The library was amplified to 4×10¹⁰ pfu/mlfor screening clones. The amplification was performed on plates bygrowing cells to saturation overnight in media containing 0.2 % maltose,then adding 10⁴ to 10⁵ pfu of library per 0.6 ml of cells and allowingthe phage to adsorb to the cells for 15 min at 37° C. The sample wasmixed with soft agar, plated, and incubated overnight at 37° C. The softagar/cells/phage layer was scraped from the confluent plates which werewashed with 4 ml SMG buffer (0.4 M NaCl, 10 mM MgSO₄, 50 mM Tris-HCl, pH7.5, 0.01% gelatin). The wash and phage agar were combined, 100 μl ofchloroform added, and the mixture incubated at 37° C. for 15 min withgentle agitation. The sample was centrifuged at 4000 xg at 4° C. for 10min twice to obtain a clear supernatant. Chloroform was added to a finalconcentration of 0.3% and the library stored at 4° C.

2) λ Charon 35 Sau3A I library

B. pertussis DNA (3×166 μg) was digested with Sau3A I (3×220 units) inthe presence of 10 mM Tris-HCl pH 7.5, 100 mM NaCl, 10 mM MgC12, 100μg/ml BSA for 1 min, 2 min, or 3 min in order to generate very largefragments of DNA. After each reaction, EDTA was added to 20 mM and then2.5 volumes of absolute ethanol added to precipitate the DNA asdescribed above. The DNA was resuspended in TNE and separated on a10-30% sucrose in gradient in TNE as described above. Fractions weretaken as before and the DNA fragment sizes visualized by agarose gelelectrophoresis. λ Charon 35 DNA 92×50 μg) was ligated to generate acircularized form before being digested with BamH I (2×20 units) in thepresence of 150 mM NaCl, 6 mM Tris-HCl pH 7.9, 6 mM MgCl2, 100 μg/ml BSAto remove the stuffer fragments. The lambda arms were purified bypelleting through an 8-20% potassium acetate gradient at 85,000 xg, for16 hr at 32° C. The Sau3A I digested DNA was ligated with the lambdaarms at 6° C. for 72 hr, then packaged into phage using a commercialkit. The phage library was propagated in E. coli LE392 cells and wastitred at approximately 1×10⁵ pfu/μg of lambda arms. The library wasamplified to 1-2×10¹⁰ pfu/ml for screening as described above.

EXAMPLE VII

This Example illustrates the screening of the B. pertussis libraries.

1) λ gtll genomic library

A 30-base oligonucleotide probe was synthesized based on the nucleotidesequence of the gene encoding PT subunit S4. The DNA was purified fromurea/acrylamide gels by uv-imaging and anion exchange chromatography onWhatman cellulose DE52. The sequence of the oligonucelotide was5'GTAGCCATGAAGCCGTATGAAGTCACCCCG3', coding for amino acids 16-25 of themature S4 protein. The oligonucleotide was 5' end-labelled in a reactionmix containing 10 μg DNA, 25 uCi [α-³² P]ATP, 4 units polynucleotidekinase in the presence of 50 mM Tris-HCl, pH 9.5, 10 mM MgC12, 5 mM DTT,5% glycerol by incubation at 37° C. for 15 min. ATP was added to 1.5 mMand the incubation continued for 1.75 hr at 37° C. 10 μg of tRNA wereadded as carrier and the labelled DNA was separated from free ATP on aSephadex G50 superfine column eluted with 0.1 M triethylammoniumbicarbonate, pH 7.6. Peak fractions were pooled and lyophilized todryness. The pellet was washed with sterile water, relyophilized thenresuspended at approximately 0.1 μg/ul.

Aliquots of the λ gtll B. pertussis genomic library were plated on aY1090 lawn on NZCYM plates containing 0.2% maltose. Plaque-lifts weremade onto nitrocellulose filters which were sequentially treated withdenaturing solution (1.5 M NaCl, 0.5 M NaOH) for 1 min, neutralizingsolution (1.5 M NaCl, 0.5 M Tris- HCl pH 8.0) for 5 min, and rinsedbriefly in 2xSSPE (0.36 M NaCl, 20 mM sodium phosphate, pH 7.4, 2 mMEDTA) before being baked at 80° C. under vacuum for 2 hr to fix the DNA.Nitrocellulose filters were subsequently incubated in a prehybridizationbuffer comprising 5xSSC (0.75M NaCl, 75 mM sodium citrate, pH 7.5), 5xDenhardt's mixture (0.1% Ficoll 400, 0.1% polyvinylpyrrolidone, 0.1%BSA), 0.1% SDS, 100 μg/ml herring sperm DNA for 2 hr at 45° C. Theprehybridization buffer was removed and fresh buffer containing 10⁷ cpmof [³² P]-labelled oligonucleotide probe was added. Hybridization wascarried out at 45° C. for 16 hr. The radioactive solution was removedand the filters rinsed briefly twice at room temperature with 5xSSC,0.1% SDS to remove unbound probe. The filters were further washed twicewith 5xSSC, 0.1% SDS for 1 hr at 50° C. then air-dried and subjected toautoradiography.

The plaque-containing plates were aligned with their autoradiograms andputative positive plaques were subjected to another two rounds ofpurification on plates. One clone (λ gtll-15-4-1) was chosen fordetailed examination by Southern blot analysis.

2) λ Charon 35 genomic library

Aliquots of the λ Charon 35 B. pertussis genomic library were plated onan LE392 lawn on NZCYM plates containing 0.2% maltose. The plaque-lift,hybridization and washing protocols were performed as described.Positive plaques were purified twice more on plates and several clones,λ Ch 35 111, 121, 411, 421 and 431, were examined by Southern blotanalysis.

EXAMPLE VIII

This Example illustrates the analysis of the genomic clones.

1) Preparation of phage DNA

One litre (2×500 ml) of phage culture was prepared. LE392 or Y1090 cellswere grown overnight in medium containing 0.2% maltose. Cells (10¹⁰)were spun down at 4400 xg for 5 min at 4° C. and the pellet resuspendedin 1 ml SMG buffer. Phage stock (1.2×10⁸ pfu) was added to the mixtureand incubated at 37° C. for 15 min to absorb the phage to the cells. Thephage/cell mixture was inoculated into 500 ml of medium and the cultureshaken vigorously at 37° C. until lysis began (4-4.5 hr). Chloroform (10ml) was added and shaking continued at 37° C. for an additional 15 minto complete the lysis. The sample was cooled to room temperature andDNase I and DNase-free RNase A (1 μg/ml each) were added for 30 min atroom temperature. The cell debris was pelleted at 3500 xg for 20 min,then 29.2 g NaCl and 50 g polyethylene glycol (PEG 6000) were added to500 ml of supernatant. The sample was gently agitated at roomtemperature to dissolve the solids, then incubated at 0° C. for 1-2 hrto precipitate the phage. The phage were harvested by centrifuging at4400 xg at 4° C. for 20 min and were resuspended in 8 ml TM buffer (50mM Tris-HCl, pH 7.5, 10 mM MgSO₄). Extraction with 8 ml chloroform toremove the PEG gave a clear supernatant which was applied to a stepgradient of 5% and 40% glycerol in TM buffer and centrifuged at 154,000xg at 4° C. for 1 hr. The supernatant was discarded leaving a phagepellet which was resuspended in 0.5 ml TM buffer. DNase I was added to 5μg/ml and RNase A to 50 μg/ml and the sample incubated at 37° C. for 30min. EDTA was added to 20 mM, pronase to 0.5 mg/ml, SDS to 0.5%, and thesample further incubated at 37° C. for 1 hr. The sample was gentlyextracted once each with phenol, phenol:chloroform 1:1, and chloroformand the phage DNA precipitated with ethanol.

2) Results

Clone 15-4-1 which was derived from the EcoR I gtll library, was foundby Southern blot analysis to contain the 4.6 kb EcoR I fragment encodingthe entire TOX gene plus small 5'- and 3'-flanking regions.

The λ Charon 35 clones were found to be closely related. Some clonescontained the entire TOX operon plus flanking regions in eitherorientation, and others did not include the entire TOX region.

The maps of clones 15-4-1, Ch 111, Ch 121/411, Ch 431 and Ch 421 areshown in FIG. 2.

EXAMPLE IX

This Example illustrates the construction of pUC-based plasmidscontaining the pertussis toxin operon (TOX) or portions thereof.

Phage DNA from the λ gtll clone 15-4-1 was prepared as described anddigested with restriction endonuclease EcoR I using standard methods.The DNA was purified by gel electrophoresis in low-melting-pointagarose. The 4.6 kb band was identified by uv-illumination of theethidium bromide stained gel and excised. The DNA was extracted by afreeze-thaw technique employing 0.3M sodium acetate, pH 7.0 and wasprecipitated with ethanol. DNA from pUC8:2, a derivative of pUC8containing two extra restriction sites for Bgl II and Xba I in itsmultiple cloning site, was digested with EcoR I. The linearized DNA wasdephosphorylated by standard methods using calf alkaline phosphatase(CAP), phenol extracted and precipitated with ethanol.

The pUC8:2-vector DNA and 15-4-1-derived-TOX DNA were ligated in astandard reaction and the ligation mixture used to transform competentJM109 cells according to standard procedures. The resulting colonieswere analysed by a rapid DNA screening technique and two clones werechosen for large-scale preparation of plasmid DNA. These clones, J-169-1and J-169-2, differed only in the orientation of the TOX insert. Theconstruction of these clones is illustrated in FIG. 3. A deposit ofplasmid J-169-1 was made with ATCC on Nov. 23, 1988 under accessionnumber 40518;

EXAMPLE X

This Example illustrates the sequencing of the TOX operon.

1) Clones used

The clone J-169-1 was used as the source for all sequencing clones. TheTOX operon was divided into five approximately equal DNA segments andwas subcloned into M13mp18, M13mp19 or pUC8:2 as illustrated in FIGS.4a, b, c, d and e.

2) Preparation of samples

M13 clones were maintained in JM101 and DNA for sequencing was preparedfrom single plaques on homogeneous plates. A saturated JM101 culture wasdiluted 1:50 with fresh medium and infected with a single plaque. Theculture was grown with vigorous shaking at 37° C. for 6 hr. The cellswere removed by centrifugation and the supernatant treated with 1/4volume of 20% PEG 6000, 2.5 M NaCl to precipitate phage. The suspensionwas centrifuged and the phage pellet was resuspended in TE, thenextracted gently twice each with phenol, phenol:chloroform (1:1) andchloroform. The phage DNA was precipitated with sodium acetate andethanol, washed with 70% ethanol and dried. The DNA was resuspended insterile water to a concentration of about 1 μg/ml for sequencing.

Sequencing primers of approximately 17-20 bases were synthesized on anABI 380A DNA synthesizer using phosphoroamidite chemistry and werepurified as described above.

3) Sequencing

The dideoxy chain termination method of Sanger was used for allsequencing reactions, employing either Klenow polymerase or Sequenase T7enzyme.

4) Results

The entire TOX operon, as previously defined, was sequenced and theresult compared with published sequences. There was excellent agreementwith the TOX sequence of strain BP 165 reported by Nicosia et al.,except for four base differences. The T at position 315 in the5'-flanking region is unique to B. pertussis strain 10536. The threeother substitutions are in the S1-coding region at positions 710, 1200and 1202 resulting in two unique amino acids, GLU34 and VAL198. Thenucleotide sequence and derived amino acid sequence are shown in FIG. 5.

EXAMPLE XI

This Example illustrates mutagenesis of the TOX gene.

1) Clones used

For mutations in the S1 gene, clone S-2403 (M13mp18/S1) was used and forthe mutations in the S3 gene, clone S-2664-5-6, (M13mp18/S3(c)) wasused. These clones are represented in FIG. 4.

2) Mutagenesis protocol

Single-stranded DNA was prepared from phage stocks derived from singleplaques on homogeneous plates as described previously. Mutagenic primersof appropriate sequence and length were synthesized on an ABI 380A DNAsynthesizer.

Commercial kits based on the phosphorothioate procedure developed byEckstein were used for in vitro mutagenesis. Briefly, the mutagenicoligonucleotide was annealed to the single-stranded (wild-type) templateand polymerization carried out using as substrates a phosphorothioatedCTP analogue and natural dATP, dGTP and dTTP. The double-stranded DNAwas nicked with Nci I and the native strand digested with exonucleaseIII beyond the point of the mutation. The complementary strand wasprotected from Nci I-nicking by the phosphorothioate groups. Thecomplementary strand then served as a template in a second round ofpolymerization, to yield double-stranded DNA with the mutation in bothstrands. This DNA was amplified in E. coli, and the mutation confirmedby sequencing.

Thirty-five primary mutations were generated and an additional 14 werederived by constructing crosses among these. The mutation efficiencyvaried with the change desired. From one to six base changes anddeletions of up to 15 consecutive bases were accomplished. The resultingamino acid changes are summarized in Table 1a.

EXAMPLE XII

This Example describes the construction of plasmids for expression ofmutated TOX genes in B. parapertussis and characterization of the PTanalogues produced.

1) Replicating plasmids

Replicative-form DNA from M13 clones was used to reconstruct the TOXoperon containing the desired mutation in pRK404. pRK404 is a derivativeof pRK290, a conjugating plasmid of the pRK2 family, incompatibilitygroup P-1. It is 10.6 kb in size, carries a tetracycline resistance(Tet^(R)) gene, and has a multiple cloning site from pUC8. Theconstruction schemes for reintegrating S1 and S3 primary mutations intothe operon are shown in FIG. 6 and the resulting clones are indicated inTable 1a. Crossed mutations in S1 were generated using internalrestriction sites, especially the unique Sal I site. A general schemefor crossed mutations in S1 is also shown in FIG. 6 and the resultingclones are indicated in Table 1a.

2) Suicide plasmids

A conjugative but non-replicative plasmid was developed for randomintegration of TOX or mutated TOX into the chromosome of Bordetellaspecies. FIG. 7 demonstrates the construction of these clones.

Plasmids of the types described in (1) and (2) above were introducedinto B. pertussis by conjugation. The resulting strains were grown inshake-flasks or in a fermentor, and the culture supernatants wereassayed as follows for concentration of toxin analogue by ELISA.Microtitre plates were coated with fetuin (2 μg/ml) in 0.05M potassiumcarbonate, pH 9.6 at 4° C. overnight in a humid environment. The plateswere then wased twice with Delbecco's PBS containing 0.1% w/v Tween-20and dried. Sample supernatants or wild-type PT were serially diluted andadded to the wells, and the plates incubated for 30 min at roomtemperature then washed. Bound PT was detected usingperoxidase-conjugated affinity-purified rabbit anti-PT antibodies.

Residual toxicity was measured by the CHO cell clustering assay, todetermine the toxicity relative to native PT. Certain PT mutants werepurified as described for native PT in Example I, and assayed forADP-ribosyltransferase activity. These data are summarized in Table 1b.Expression of the S1 epitope recognized by MAb PS21 was assessed by amodified indirect ELISA on culture supernatants. Fetuin-bound PTanalogues were reacted with PS21 as the first antibody and visualizedwith an enzyme-conjugated affinity-purified goat anti-mouse IgG as thesecond antibody. The presence or absence of the S1 epitope recognized byMAb PS21 is indicated in Table 1b.

EXAMPLE XIII

This Example illustrates the construction of plasmids for deletion andreplacement of the endogenous B. pertussis TOX operon.

1) Plasmids containing TOX flanking regions a) 5'-flanking region

The Ch 421 DNA was first digested with Bgl II and and 11 kb fragment waspurified by agarose gel electrophoresis. The Bgl II fragment. wasdigested with Xma I and the 5 kb band subcloned into pUC8:2 previouslyrestricted with Xma I and dephosphorylated. JM109 cells were transformedwith the ligation mixture to give colonies which were analysed by arapid DNA screening method. The clone J-183-9 was found to containapproximately 2.9 kb of the 5'-flanking region, the TOX promoter and thegenes for subunits S1 and S2. FIG. 8a shows the derivation of cloneJ-183-9.

b) 3'-flanking region

The Ch 111 DNA was digested with Sal I and an approximately 8 kbfragment of B. pertussis DNA was gel-purified. This DNA fragment wasinserted into pUC8:2 previously digested with Sal I anddephosphorylated. JM109 transformants were screened and the cloneJ-219-111-3 was identified as containing part of the S1 gene, all of theremaining structural genes, and about 3.9 kb of the 3' flanking region.FIG. 8b shows the construction of this clone.

TOX gene with 5'- and 3'-flanking regions.

Clone J-183-9 was digested with Xba I and the approximately 7 kbfragment containing pUC8:2, the 5'-flanking region and the promoterregion of the S1 gene was gel-purified and dephosphorylated. J-219-111-3DNA was. digested with Xba I and the approximately 8 kb fragmentcontaining the structural genes for subunits S2 to S5 and the3'-flanking regions were gel- purified. These DNA fragments were ligatedand the JM109 transformants were screened to give clone J-229-17. Thisclone contains about 2.9 kb of the 5'-flanking sequence, the entire TOXoperon, and about 4 kb of the 3'-flanking sequence. Its construction isillustrated in FIG. 8c. (Plasmid J-229-17 was deposited with ATTC onNov. 30, 1989 under accession number 40,715)

2) TOX-deleting plasmids

Plasmid S-2832-5 contains the Tet^(R) gene from plasmid pRK404 and itsconstruction is shown in FIG. 9. The Tet^(R) gene was cloned as an EcoRI/BamH I restriction fragment into plasmid pN01523 to generate pGZ62.Plasmid pGZ63 contains the 5'- and 3'-flanking regions without anyintervening DNA. The S12-Tet^(R) gene-sandwich from pGZ62 was clonedbetween the flanking regions of pGZ63 to produce plasmid pGZ65. Theconstruction of these plasmids is summarized in FIG. 8d.

3) TOx-reintegrating plasmids

To express mutated TOX genes in TOX⁻ strains of B. pertussis conjugativesuicide plasmids of the type shown in FIG. 10 were constructed. Theycontain the TOX gene, extensive 5'- and 3'-flanking sequences and have aTet^(R) gene for selection cloned downstream from the TOX codingregions.

EXAMPLE XIV

This Example illustrates the deletion of the TOX gene from the B.pertussis chromosome and the reintegration of in vitro-mutated TOXgenes.

1) Transformation of B. pertussis

Strains of B. pertussis were transformed by electroporation. Cells weregrown in 100 ml of modified Stainer-Scholte medium to a density of about109 cells/ml, harvested in a clinical centrifuge (4000 xg for 15 min at20° C.), washed in 25 ml of electroporation buffer (0.3M sucrose, 1 mMMgC12, 7 mM potassium phosphate, pH 7.2) and resuspended in 10 ml of thesame. Plasmid DNA was added to 500 μl of the cell suspension and themixture incubated on ice for 10 min. The cells were subjected to asingle 25 kV/cm, 40 us exponential decay voltage pulse with a BTXTransfector 100, using a cuvette electrode with a 0.8 mm gap. Three mlof medium were added and the cells incubated with shaking at 37° C. for60 min. The cells were harvested by centrifugation at 12,000 xg for 2min, resuspended in 100 μl of medium, spread onto a Bordet-Gengou platewith antibiotic selection and incubated for 2-5 days at 37° C.

a) Deletion and replacement of the TOX operon

B. pertussis str29 is a spontaneous rpsL streptomycin resistantderivative of B. pertussis 10536. (A deposit of B. pertussis strainstr29 was made with ATCC on Nov. 30, 1989 under accession no. 53972)Plasmid pGZ65 contains a gene cartridge consisting of the pRK404 Tet^(R)gene and the E. coli S12 gene cloned between the 5'- and 3'-flankingsequences of the TOX operon. This plasmid was linearized with Hind IIIand used to transform B. pertussis str29 to Tet^(R), str6 resulting inthe deletion of the TOX operon :by homologous recombination. ThisTOX-deleted strain was termed 29-8 and a Southern blot analysisdemonstrated the excision of the TOX allele and its replacement by theTc^(r), S12 gene cartridge. This strain was deposited with ATCC on Nov.30, 1989 under accession number 53,973. To excise the S12-Tet^(R) genecartridge, strain 29-8 was subsequently transformed with linear pGZ63plasmid DNA. Plasmid pGZ63 consists of the TOX 5'- and 3'-flankingsequences but contains no intervening DNA. Transformation with thisplasmid resulted int he generation of B. pertussis 29-9 which is astreptomycin-resistant, TOX-deleted strain but contains no heterologousDNA inserted at the TOX locus. This strain was used as the host forexpression of in vitro mutated TOX genes. Plasmids of the type shown inFIG. 10 contain a gene cartridge consisting of a mutated TOX gene and aTet^(R) gene. This gene cartridge was recombined into the B. pertussis29-9 chromosome following introduction of the plasmid into the strain byconjugation or transformation. Expression of the TOX gene, toxicity ofthe PT analogues and maintenance of the S1 epitope recognised by MAbPS21 were determined as described before. The recombinant B. pertussisstrains constructed and the properties of the secreted PT analogues areshown in Table 5.

EXAMPLE XV

This Example describes the in vivo testing of PT mutants in mice.

PT mutants were purified from culture supernatants and recombinant B.parapertussis strains as indicated in Example I. These proteins wereinjected into mice at three different doses to test the followingcharacteristics, according to standard procedures: acute toxicity,histamine sensitization activity and potency int he mouse intracerebralchallenge test. The results are presented in Table 2.

To test their immunogenicity, PT analogues were injected into femaleBALB/C mice, 9 to 11 weeks old, at doses of 2.0, 0.5 and 0.125 μg. Micewere pre-bled and immunized on day 0. On day 23 the mice were bled againand boosted with the same immunogen, and on day 37 the mice were bledagain. Blood samples (0.4-0.5 ml/mouse) were collected by orbital sinusbleeding and the resulting sera stored at -20° C. to await testing. Serawere assayed for their ability to neutralize PT-induced CHO cellclustering (Table 3), and for specific antibody responses inantigen-coat, indirect ELISA (Table 4). As may be seen form Tables 3 and4, PT analogues are capable of inducing neutralizing antibodies andanti-PT, anti-S1 and anti-B oligomer responses.

SUMMARY OF DISCLOSURE

In summary of this disclosure, the present invention provides a novelmethod of detoxifying pertussis by identification of specific functionalsites of pertussis toxin and production of recombinant holotoxins bysite-directed mutagenesis of the toxin gene. The resulting toxinanalogues are detoxified, retain an immunodominant S1 epitope, areimmunogenic and are protective against the disease pertussis.Modifications are possible within the scope of this invention.

                                      TABLE Ia                                    __________________________________________________________________________    Summary of Mutations introduced into Pertussis Toxin                          Mutation                                                                      Number                                                                              Mutation                 Clone No.                                      __________________________________________________________________________    1.    ARG.sup.9 → Δ.sup.9                                                                       S-2679-1-11                                    2.    ARG.sup.9 → GLU.sup.9                                                                           S-2815-1-8                                     3.    ARG.sup.9 → LYS.sup.9                                                                           S-2953-21                                      4.    ARG.sup.9 → HIS.sup.9                                                                           S-3046-4                                       5.    ARG.sup.13 → Δ.sup.13                                                                     S-2679-2-1                                     6.    ARG.sup.13 → GLU.sup.13                                                                         S-2779-2-1                                     7.    ARG.sup.9 -ARG.sup.13 → Δ.sup.9-13                                                        S-2829-2-19                                    8.    ARG.sup.9 ARG.sup.13 → GLU.sup.9 GLU.sup.13                                                     S-2779-3-2                                     9.    ARG.sup.58 → GLU.sup.58                                                                         J-444-2-2                                      10.   ARG.sup.57 ARG.sup.58 → Δ.sup.57 Δ.sup.58                                           J-482-11                                       11.   TYR.sup.26 → ALA.sup.26                                                                         S-3123-2                                       12.   TYR.sup.26 → CYS.sup.26                                                                         S-3140-22                                      13.   CYS.sup.41 → ALA.sup.41                                                                         S-2515-5-10                                    14.   CYS.sup.41 → SER.sup.41                                                                         S-3124-6                                       15.   CYS.sup.201 → ALA.sup.201                                                                       S-2679-3-4                                     16.   GLU.sup.129 → Δ.sup.129                                                                   S-2589-6                                       17.   GLU.sup.129 → GLY.sup.129                                                                       S-2515-3-6                                     18.   GLU.sup.129 → GLN.sup.129                                                                       S-2515-1-2                                     19.   GLU.sup.129 → ASP.sup.129                                                                       S-2515-2-4                                     20.   GLU.sup.129 → ASN.sup.129                                                                       S-2852-1-18                                    21.   GLU.sup.129 → LYS.sup.129                                                                       S-2515-4-11                                    22.   GLU.sup.129 → ARG.sup.129                                                                       M-32-2-4                                       23.   GLU.sup.129 → HIS.sup.129                                                                       S-2937-1-2                                     24.   GLU.sup.129 → PRO.sup.129                                                                       S-2959-2-28                                    25.   GLU.sup.129 → CYS.sup.129                                                                       J-478-5                                        26.   GLU.sup.129 → GLY.sup.129 II                                                                    J-4-l8-1                                       27.   GLU.sup.129 GLN.sup.129 II                                                                             J-412-9                                        28.   TYR.sup.130 → Δ.sup.130                                                                   S-2852-2-1                                     29.   TYR.sup.130 → PHE.sup.130                                                                       S-2836-15                                      30.   GLU.sup.129 TYR.sup.130 → GLY.sup.129 ALA.sup.130                                               S-2679-4-3                                     31.   GLU.sup.129 TYR.sup.130 → GLN.sup.129 ALA.sup.130                                               M-38-1                                         32.   GLU.sup.129 TYR.sup.130 → GLY.sup.129 PHE.sup.130                                               J-444-1-6                                      33.   (S3)LYS.sup.10 → GLN.sup.10                                                                     S-2995-1-2                                     34.   (S3)TYR.sup.92 LYS.sup.93 → ASN.sup.92 ARG.sup.93                                               S-2995-2-1                                     35.   (S3)LYS.sup.105 → ASN.sup.105                                                                   S-2995-3-1                                     36.   CYS.sup.41 CYS.sup.201 → ALA.sup. 41 ALA.sup.201                                                S-2818-1                                       37.   CYS.sup.41 GLU.sup.129 → ALA.sup.41 GLY.sup.129                                                 S-2549-2                                       38.   ARG.sup.9 GLU.sup.129 → GLU.sup.9 GLY.sup.129                                                   S-2966-1-5                                     39.   ARG.sup.9 GLU.sup.129 → GLU.sup.9 GLN.sup.129                                                   S-2967-1-1                                     40.   ARG.sup.9 GLU.sup.129 → GLU.sup.9 ARG.sup.129                                                   M-45-1                                         41.   ARG.sup.9 GLU.sup.129 TYR.sup.130 → GLU.sup.9 GLY.sup.129              ALA.sup.130              S-2956-1                                       42.   ARG.sup.13 GLU.sup.129 → GLU.sup.13 GLY.sup.129                                                 S-2966-2-13                                    43.   ARG.sup.13 GLU.sup.129 → GLU.sup.13 GLN.sup.129                                                 S-2967-2-17                                    44.   ARG.sup.13 GLU.sup.129 TYR.sup.130 → GLU.sup.13 GLY.sup.129            ALA.sup.130              S-2961-1                                       45.   ARG.sup.9 GLU.sup.129 → Δ.sup.9 GLN.sup.129                                               S-2730-1-1                                     46.   ARG.sup.9 GLU.sup.129 TYR.sup.130 → Δ.sup.9                      GLY.sup.129 ALA.sup.130  S-2730-3-2                                     47.   ARG.sup.13 GLU.sup.129 → Δ.sup.13 GLN.sup.129                                             S-2730-2-1                                     48.   ARG.sup.13 GLU.sup.129 TYR.sup.130 → Δ.sup.13                    GLY.sup.129 ALA.sup.130  S-2730-4-1                                     49.   GLU.sup.129 → GLY.sup.129                                                                       S-3050-1                                             (S3)TYR.sup.92 LYS.sup.93 (S3)ASN.sup.92 ARG.sup.93                     50.   Wild Type                S-2505-4-5                                     __________________________________________________________________________     Amino acid numbering corresponds to positions in the native subunits (FIG     5)                                                                            All mutations are in subunit S1 unless specified as being in S3 (S3)          II denotes use of an alternative codon                                        Δ denotes deleted residue(s)                                            Wild type refers to PT expressed from the unmutated TOX operon in B.          parapertussis.                                                           

                  TABLE 1b                                                        ______________________________________                                        In vitro characterization of pertussis toxin analogues                        obtained from recombinant B. parapertussis.                                   Mutation Residual     ADPR                                                    Number   Toxicity     Activity S1 Epitope                                     ______________________________________                                        1.       0.2          ND       -                                              2.       0.1          0.2      +/-                                            3.       0.1          ND       ++++                                           4.       0.2          0.1      +++                                            5.       0.3          ND       -                                              6.       5.0          ND       ++++                                           7.       0.4          0.1      -                                              8.       0.1          0.9      -                                              9.       0.7          0.6      +++                                            10.      0.4          ND       -                                              11.      0.5          ND       +                                              12.      6.0          ND       ND                                             13.      0.3          0.4      -                                              14.      1.4          ND       ND                                             15.      0.2          0.1      -                                              16.      0.1          ND       ++                                             17.      0.1          0.3      ++++                                           18.      0.02         0.1      +/-                                            19.      0.7          2.5      ++                                             20.      0.1          0.3      ++                                             21.      0.3          0.2      -                                              22.      0.1          ND       -                                              23.      0.2          ND       -                                              24.      0.2          ND       +                                              25.      0.4          ND       -                                              26.      0.1          0.3      ++++                                           27.      0.02         0.1      +/-                                            28.      0.2          0.1      -                                              29.      12.0         ND       ++ ++                                          30.      0.2          0.6      -                                              31.      0.4          ND       -                                              32       1.0          ND       ++++                                           33.      100          ND       ++++++                                         34.      50           100      ++++                                           35.      20           ND       ++++                                           36.      0.2          0.1      -                                              37.      0.1          0.1      -                                              38.      0.1          0.1      -                                              39.      0.1          ND       -                                              40.      0.1          ND       -                                              41.      0.2          ND       -                                              42.      0.5          ND       -                                              43.      3.0          ND       -                                              44.      0.3          ND       -                                              45.      0.4          ND       -                                              46.      0.2          0.1      -                                              47.      0.5          ND       -                                              48.      0.4          0.3      -                                              49.      0.2          0.1      ++++                                           50.      100          100      +++++                                          ______________________________________                                         Residual toxicity is the ratio of the apparent PT concentration determine     by the CHO cell clustering assay to the actual concentration of PT mutant     determined by ELISA as expressed a percentage.                                ADPR activity is the extent of ADPribosylation of bovin transducin            catalysed by a PT analogue, relative to that catalysed by an equal            concentration of wildtype PT, expressed as a percentage.                      S1 epitope refers to the expression of an immunodominant S1 epitope           recognized by a specific monoclonal antibody PS21, as compared with the       wildtype PT (+++++).                                                          ND denotes not determined.                                               

                  TABLE 2                                                         ______________________________________                                        Biological Activity of PT mutants in mice                                                    Acute     HS                                                                  Toxicity  Activity  M.P.T.                                     Analogue       LD.sub.50 (ug)                                                                          LD.sub.50 (ug)                                                                          ED.sub.50 (ug)                             ______________________________________                                        Native         2         ˜0.2                                                                              ˜2                                   GLY.sup.129    >5        ˜3  2                                          GLN.sup.129    >16       >3        16                                         ASN.sup.129    >5        ˜3  1.5                                        GLU.sup.58     >5        1.5       8.5                                        LYS.sup.9      0         6         2                                          GLY.sup.129 (S3)ASN.sup.92 ARG.sup.93                                                        >20       7         2                                          (S3)ASN.sup.92 ARG.sup.93                                                                    3         0.4       ≧2                                  ______________________________________                                         HS Activity denotes histamine sensitizing activity.                           M.P.T. denotes mouse intracerebral challenge protection test.                 LD.sub.50 is the dose resulting in death of 50% of the test animals.          ED.sub.50 is the dose resulting in protection of 50% of the test animals.     Native denotes PT from B. pertussis 10536.                               

                  TABLE 3                                                         ______________________________________                                        Neutralizing effect of immune sera on PT-induced CHO cell                     clustering                                                                    Analogue                                                                      Dose (ug)                                                                              Pre-bleed  Post-1 bleed                                                                             Post-2 bleed                                   ______________________________________                                        GLY.sup.129                                                                   2.0      <2         <2         256                                            0.5      <2         <2         128                                            0.125    <2         <2          64                                            GLN.sup.129                                                                   2.0      <2         <2         128                                            0.5      <2         <2         256                                            0.125    <2         <2         128                                            ASN.sup.129                                                                   2.0      <2         <2         512                                            0.5      <2         <2         128                                            0.125    <2         <2         256                                            Saline   <2         <2         <2                                             ______________________________________                                         Mice were prebled and immunized on day 0. On day 23 they were bled again      (post1 bleed) and boosted. Final sera were obtained on day 37 (post2          bleed).                                                                       The neutralizing ability of the sera is expressed as the maximum dilution     at which CHO cell clustering was inhibited.                              

                  TABLE 4                                                         ______________________________________                                        Specific antibody titres of immune sera                                       Analog                                                                        Dose   Pre-bleed   Post-1 bleed Post-2 bleed                                  (ug)   PT  S1   B      PT   S1   B    PT   S1   B                             ______________________________________                                        GLY.sup.129                                                                   2.0    NR NR    NR     63   2    50   500  80   200                           0.5    NR NR    NR     13   1    8    160  32   56                            0.125  NR NR    NR     10   0.5  8    200  32   80                            GLN.sup.129                                                                   2.0    NR NR    NR     22   0.7  20   200  40   125                           0.5    NR NR    NR     8    0.5  6    200  40   100                           0.125  NR NR    NR     5    <0.5 2    125  20   50                            ASN.sup.129                                                                   2.0    NR NR    NR     40   1    40   500  140  280                           0.5    NR NR    NR     7    <0.5 3    316  22   80                            0.125  NR NR    NR     7    <0.5 4    180  63   125                           Saline NR NR    NR     NR   NR   NR   NR   NR   NR                            ______________________________________                                         Immunization and bleeding were performed as described in Table 3.             Antigens used were PT holotoxin, isolated S1 subunit and isolated B           oligomer.                                                                     The units are the dilution factor divided by 1000 giving an ELISA             absorbance value equal to twice the background.                               NR denotes not reactive with antigen.                                    

                  TABLE 5                                                         ______________________________________                                        In vitro characterization of pertussis toxin analogues from                   recombinant B. pertussis                                                      Mutation          Residual  ADPR    S1                                        Number Clone      Toxicity  Activity                                                                              Epitope                                   ______________________________________                                        9      S-3036-2   0.2       0.3     +++                                       13     S-3122-3-1 0.1       ND      ND                                        17     S-2962-1-2 0.2       ND      ND                                        18     S-2962-2-1 0.1       ND      ND                                        34     S-3122-3-1 50        ND      +++++                                     49     S-3122-2-3 0.1       ND      +++                                       50     S-3006-3   100       100     ++++                                      ______________________________________                                         All terms are as defined in Tables 1a and 1b.                                 ND denotes not determined.                                               

What we claim is:
 1. A strain of Bordetella capable of expressing animmunoprotective genetically-detoxified mutant of pertussis holotoxinand in which the toxin operon has been replaced by a mutant operonformed by mutagenesis of a nucleotide sequence coding for at least onespecific amino acid residue which contributes to pertussis toxintoxicity.
 2. The strain of claim 1 which is selected from the groupconsisting of B. pertussis strain S-2962-1-2 having ATCC accessionnumber 53837, B. pertussis strain S-2962-2-1 having ATCC accessionnumber 53836, B. pertussis strain S-3036-2 having ATCC accession number53835, B. pertussis strain S-3122-3-1 having ATCC accession number51834, and B. pertussis strain S-3122-2-3 having ATCC accession number53833.
 3. A method for the production of an immunoprotective,genetically-detoxified mutant of pertussis holotoxin, whichcomprises:identifying at least one amino acids residue of the holotoxinwhich contributes to toxicity of the holotoxin, effecting mutagenesis ofthe holotoxin operon to remove or replace a nucleotide sequence codingfor said at least one such amino acid residue and to produce a mutatedholotoxin operon, substituting the mutated holotoxin operon for thenative operon in the Bordetella organism, and growing the transformedorganism to produce the immunoprotective, genetically-detoxifiedholotoxin.
 4. The method of claim 3 wherein said at least one amino acidresidue is identified as a single functionally-active amino acid in theS1 subunit, and said mutagenesis is effected at the codon for saidsingle amino acid.
 5. The method of claim 4 wherein said single aminoacid is GLU¹²⁹ or ARG⁵⁸.
 6. The method of claim 3 wherein said at leastone amino acid residue is identified as multiple functionally-activeamino acids, and said mutagenesis is effected at codons for each saidamino acid.
 7. The method of claim 6 wherein said multiple amino acidscomprise GLU¹²⁹ TYR¹³⁰ or GLU¹²⁹ /(S3)TYR⁹² LYS⁹³.
 8. The method ofclaim 3 wherein said substitution step is effected by conjugation or byhigh voltage electroporation.